We have previously described a single morphogenesis pathway for the Bacillus subtilis bacteriophage phi29 in which the 12 megadalton genome is packaged into a relatively simple prohead constructed with the aid of a recyclable core-scaffolding protein. The overall objective of the present proposal is to construct a model of phi29 that is consistent with the events that occur during assembly and to reproduce in vitro the events that lead to prohead formation and DNA-encapsidation. Specifically, we will redetermine the mass of the phi29 virion and the number of copies of each type of structural protein, and we will seek nonprotein structural components. We will study the origin, location and function of a multifunctional protein that links the ends of phi29 DNA in the virion. We will determine the stoichiometry and location of the 3-4 proteins comprising the relatively unstable prohead and the 2-3 proteins of the first stable DNA-containing intermediates. We will investigate the role of the phi29 prohead by studying DNA encapsidation in vitro, and we will attempt in vitro assembly of functional proheads from partially purified protein constituents. Finally, we will continue fine structure mapping of the cistron specifying the major head protein in an effort to correlate mutations affecting specific protein-protein interactions in head morphogenesis with map position and ultimately amino acid sequence.